1. Field of the Invention
The claimed subject matter pertains to reconstituted stable human serum based controls and/or calibrants used to assist in the measurement of total protein, total CK, CK isoenzymes, total LD, LD isoenzymes, and specific proteins. In particular, the claimed subject matter pertains to a lyophilized stable human serum based control with long term shelf life and relatively long term reconstituted stability.
2. Description of the Related Art
A number of physiological conditions and states are correlated with the above enzymes. An elevated total CK level correlates with myocardial infarction, myocardial ischemia, stenocardia, tachycardia, myocarditis, subarachnoid hemorrage, stroke, brain tumor, convulsions, meningitis, encephalitis, acute psychosis, epilepcy, muscular dystropy, viral myositis, severe myoglobinurea, malignant hyperthermia, carbon monoxide poisioning, etc.
An increased level of the principle isoenzyme CK-MB is associated with myocardial infarction, myocardial ischemia, muscular dystrophy, myositis, severe myoglobinurea, malignant hyperthermia and carbon monoxide poisoning. Increased levels of CK-MM has no current clinical significance. The presence of LD, together with CK-MB, is quite specific for myocardial infarction. The isoenzymes of LD are also correlated with certain disease states.
Tests have been devised to determine the presence or concentration of these enzymes in bodily fluids. See e.g. NHL CK MB, NHL CK, NHL LD (DR 5020), ACA.TM. LDH pack, Paragon.TM. (Beckman) CK isoenzyme electrophoresis, etc.
In order to maintain the precision and accuracy of these tests, good laboratory practice dictates that control materials be included with patient samples each time an assay is run. If control values appear out of range, if upward or downward trends are noted, or if sudden shifts are seen in control values, all operating parameters, including instrument calibration, should be checked.
It has been observed, however, that CK and LD enzymes are not highly stable. In order to facilitate transport and storage of these tests the controls are lyophilized. It has also been observed that enzyme activity can also be lost during lypholization and reconstitution.
The enzyme CK, by virtue of its reactive SH group, is known to be one of the least stable enzymes. In order to preserve the enzyme activity of CK and other SH-- containing enzymes one or more thiol-compounds are added to the CK solution. N. Kar, "Activation of Creatine Phosphokinase by Sulfydryl Compounds in Normal and Muscular Dystrophy Sera," 18 Proc. Exp. Bio. Med. 662, 663 (1985). D. Miyada, "Creatine Kinase Reactivation by Thiol Compounds", 58 Clinica Chimica Acta 97 (1975); G. Szasz, "Creatine Kinase in Serum: 5. Effect of Thiols on Isoenzyme Activity During Storage at Various Temperatures", 24 Clin. Chem. 1557 (1978). In addition to generally stabilizing CK, it is often desirable to lyophilized and reconstitute solutions containing this enzyme. A number of researchers suggest that reactivation should occur in the presence of added thio activator. O. Hetland, "Activation of Creatine Kinase Activity in Lyophilized Control Materials", 37 Scand. J. Clin. Lab. Invest. 563 (1977); L. Morin, "Creatine Kinase: Stability, Inactivation, Reactivation", 23 Clin. Chem. 646 (1977).
Another approach to stabilizing SH enzymes is through modification of the reactive --SH groups by some reagent. These reagents, e.g. iodoacetate, lead to irreversible reaction of the SH group and an undesirable loss of enzyme activity.
Still another approach to stabilizing SH enzymes involves reacting an organodisulfide, organothio sulfonate, tetrathionate or a mixture thereof with CK to form a stable intermediate composition for incorporating into a diagnostic reference standard. See U.S. Pat. No. 4,339,533.
In addition, it has been observed that salts and coenzymes can protect certain enzymes against denaturation. DiSabato studied the action of a number of organic and inorganic salts on the inactivation of chicken lactic dehydrogenase by urea. G. DiSabato, "The Denaturation of Lactic Dehydrogenases", 240 J. Biological Chem. 1072, 1073 (1965). It has also been observed that stability varies depending on the pH of the solution. C. Chervenka, "The Urea Denaturation of Chymotrypsinogen as Determined by Ultraviolet Spectral Changes; The Influence of pH and Salts", 82 J. Am. Chem. 582 (1960). In addition, it is known that the stability, as measured by a decrease in activity, of individual enzymes, varies as a function of time and storage temperature. P. Hissin et al., "Stability of Total Dehydrogenase (LD) and LD isoenzymes at Different Storage Temperatures as a Function of Time", 31 Clin. Chem. 999 (1985); H. Kreutzer "Lactic Dehydrogenase Isoenzymes in Blood Serum After Storage at Different Temperatures", 9 Clin. Chim. Acta 64 (1964).